Transcriptional Regulatory Complexes in Epidermal Differentiation Project Summary My long-term career goal is to lead a productive academic group at a research university, advancing the field of epithelial biology, and serving as a mentor to young scientists. My scientific interests lie in understanding the complex biology behind transcriptional regulatory networks mediating epidermal homeostasis. As I build a solid research program in this area, my immediate goals are to obtain mentorship in new areas, including epigenomics, bioinformatics, and proteomics, which will greatly enhance my current expertise in genetics, biochemistry and molecular biology. In addition to the valuable expertise provided by my mentor, six additional faculty members at Stanford University with expertise in these areas have agreed to serve as collaborators for the proposed studies. Furthermore, I will acquire additional knowledge through limited didactic courses, and participation in leadership and management seminars offered at Stanford will further prepare me for my transition to independence. Altogether, these efforts will allow me to expand my current research to provide a platform for establishing a new research group and aid in my transition to independent investigator. The goal of this K01 proposal is to investigate the transcriptional regulatory complexes involved in epidermal differentiation. I have recently identified the MAF/MAFB transcription factors (TFs) as critical regulators of epidermal homeostasis mediating progenitor cell cycle exit and terminal differentiation. These TFs are themselves regulated by long non-coding RNAs and mediate downstream activation of important epidermal TFs in cooperation with p63, the master regulator of epidermis. Furthermore, our analysis revealed MAF/MAFB localized to putative enhancers at the genomic level. Due to their ability to both suppress progenitor genes and activate terminal differentiation genes, we believe MAF/MAFB exist simultaneously in activating and repressive complexes. Thus, the aims are to 1) Characterize the epigenomic effects of MAF/MAFB during epidermal differentiation and, 2) Functionally characterize the MAF/MAFB protein interactome to understand how MAF/MAFB function in activating and repressive complexes. We will begin by determining the epigenomic effects of MAF/MAFB in Aim I. The contribution of MAF/MAFB to chromatin accessibility and genome architecture will be addressed in the context of MAF/MAFB CRISPR-mediated knockout keratinocytes. We will use ATAC-seq to identify changes in chromatin accessibility comparing MAF/MAFB loss to control differentiated keratinocytes. To assess changes in genome shape, we will interrogate promoter:DNA contacts using capture Hi-C in control vs MAF/MAFB knockout keratinocytes that have undergone differentiation. These unbiased approaches will provide genome-wide data for characterizing the epigenome during epidermal differentiation, assessing the impact of MAF/MAFB loss, and identifying critical genomic regulatory elements required for differentiation. Furthermore, characterization of regulatory elements will allow for subsequent identification of additional factors, or potential co-regulators, involved in MAF/MAFB mediated gene regulation. We will first test candidate co-regulators already identified through FOCIS analysis of MAF/MAFB bound loci and then incorporate results from epigenomic studies to identify and characterize putative co-regulators. In Aim II, we will focus on functionally characterizing the MAF/MAFB protein interactome. Because of their ability to suppress progenitor genes and activate differentiation genes, we hypothesized that MAF/MAFB exist simultaneously in activating and repressive complexes to mediate gene expression. We will first characterize the MAF/MAFB interactome using tandem affinity purification and a proximal protein biotinylation (BioID) approach coupled with mass spectrometry to identify proteins associating with MAF/MAFB during epidermal differentiation. These approaches will identify stable and transient MAF/MAFB interactors. To functionally characterize candidate MAF/MAFB co-regulators, we have developed a CRISPR/Cas9 screen to identify factors required for terminal differentiation. Candidates that arise from this screen will be validated by gene expression profiling and in tissue models. Taken together, this aim will serve to functionally characterize MAF/MAFB activating and repressive complexes operating during epidermal differentiation. Over the course of the training period, I plan to have expanded my expertise into the areas of epigenomics, bioinformatics, and proteomics, all while characterizing the complexity of epidermal transcriptional regulation. The findings arising from this proposal will inform the skin biology community of critical DNA regulatory elements required for human epidermal differentiation, how MAF/MAFB participates in functional regulation of these elements, and what additional factors are critical for epidermal differentiation. These findings may form the foundation of future treatment strategies for disorders characterized by disrupted epidermal homeostasis.